NANOSECOND AND MICROSECOND TIME-RESOLVED FTIR SPECTROSCOPY OF THE HALORHODOPSIN PHOTOCYCLE

Step-scan Fourier transform infrared spectroscopy with 50 ns time reso lution was applied to the early stages of the photocycle of halorhodop sin (hR) for the temperature range 3-42 degrees C. Kinetic data analys is with global fitting revealed two distinct kinetic processes associa ted with relaxations of the early red-shifted photoproduct hK; these p rocesses have time constants tau(1) similar or equal to 280 ns and tau (2) similar or equal to 360 mu s at 20 degrees C. Spectral features de monstrate that the tau(1) process corresponds to a transition between two distinct bathointermediates, hK(E) and hK(L). The vibrational diff erence bands associated with both tau(1) and tau(2) transitions are sp read throughout the whole 1800-900 cm(-1) range. However, the largest bands correspond to ethylenic C=C stretches, fingerprint C-C stretches and hydrogen out-of-plane (HOOP) wags of the retinal chromophore. The time evolution of these difference bands indicate that both the tau(1 ) and tau(2) decay processes involve principally a relaxation of the c hromophore and its immediate environment. The decay of the intense HOO P vibrations is nearly equally divided between the tau(1) and tau(2) p rocesses, indicating a complex chromophore relaxation from a twisted n onrelaxed conformation in the primary (hK(E)) bathointermediate, to a less-twisted structure in hK(L), and finally to a roughly planar struc ture in the hypsochromically shifted hL intermediate. This conclusion is also supported by the unexpectedly large positive entropy of activa tion observed for the tau(1) process. The two relaxations from hK(E) t o hL are largely analogous to corresponding relaxations (K-E --> K-L - -> L) in the bacteriorhodopsin photocycle, except that the second step is slowed down by over 200-fold in hR.